Chromatographic purification of the plasmin-like enzyme from clamworm (Perinereis aibuhitensis Grub) and its fibrinolytic activity by metal ions.

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.

2023 AHA/ACC/ACCP/ASPC/NLA/PCNA Guideline for the Management of Patients With Chronic Coronary Disease: A Report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines.

AIM: The "2023 AHA/ACC/ACCP/ASPC/NLA/PCNA Guideline for the Management of Patients With Chronic Coronary Disease" provides an update to and consolidates new evidence since the "2012 ACCF/AHA/ACP/AATS/PCNA/SCAI/STS Guideline for the Diagnosis and Management of Patients With Stable Ischemic Heart Disease" and the corresponding "2014 ACC/AHA/AATS/PCNA/SCAI/STS Focused Update of the Guideline for the Diagnosis and Management of Patients With Stable Ischemic Heart Disease." METHODS: A comprehensive literature search was conducted from September 2021 to May 2022. Clinical studies, systematic reviews and meta-analyses, and other evidence conducted on human participants were identified that were published in English from MEDLINE (through PubMed), EMBASE, the Cochrane Library, Agency for Healthcare Research and Quality, and other selected databases relevant to this guideline. STRUCTURE: This guideline provides an evidenced-based and patient-centered approach to management of patients with chronic coronary disease, considering social determinants of health and incorporating the principles of shared decision-making and team-based care. Relevant topics include general approaches to treatment decisions, guideline-directed management and therapy to reduce symptoms and future cardiovascular events, decision-making pertaining to revascularization in patients with chronic coronary disease, recommendations for management in special populations, patient follow-up and monitoring, evidence gaps, and areas in need of future research. Where applicable, and based on availability of cost-effectiveness data, cost-value recommendations are also provided for clinicians. Many recommendations from previously published guidelines have been updated with new evidence, and new recommendations have been created when supported by published data.

Exercise testing unmasks exaggerated blood pressure independent of fibrinolytic response in Black but not White postmenopausal females.

Exercise testing unmasks more exaggerated systolic blood pressure responses (SBP) in Black compared with White male adults. Such responses, if translatable to females, may detect racial disparities particularly relevant during menopause. Given the endothelial involvement in BP regulation and as a source of fibrinolytic markers, it follows that fibrinolytic and BP response to exercise could be linked. Thus, we examined BP and fibrinolytic responses to exercise testing in Black and White postmenopausal females. Postmenopausal females (Black = 40; White = 41; 51-70 yr) performed maximal treadmill exercise. BP and blood draws were conducted before and immediately after exercise. Plasma samples, using minimal stasis, were analyzed for tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) activity and antigen, respectively. Resting SBP and fibrinolytic potential were similar between races. Black females exhibited greater increases in SBP during exercise [change (d)=75, 95% CI: 64-86 mmHg, P < 0.001] than White females (d = 60, 95% CI: 48-71 mmHg, P < 0.001). Black compared with White females had smaller changes in tPA (d = 3.27, 95% CI: 2.28-4.27 IU/mL, P < 0.001 vs. d = 5.55, 95% CI: 4.58-6.53, P < 0.001) and PAI-1 (d = -2.89, 95% CI: -4.39 to -1.40 IU/mL, P < 0.001 vs. d = -5.08, 95% CI: -6.59 to -3.61, P < 0.001) activities after exercise. SBP exercise-induced changes were not associated with tPA (r = -0.10, P = 0.42) or PAI-1 (r = 0.13, P = 0.30), without any influence of race (P > 0.05). Our findings show that maximal exercise unmasks risk factors for cardiovascular disease in Black postmenopausal females.NEW & NOTEWORTHY Exaggerated SBP responses to exercise testing are more frequent in Black than in White male adults. Such responses, if translatable to females, may detect early racial disparities arriving during menopause. Because the endothelium regulates BP and fibrinolytic responses, these could be linked during exercise. At peak exercise, Black but not White postmenopausal females had more exaggerated SPB responses regardless of reduced fibrinolytic potential. Maximal exercise unmasked risk factors for cardiovascular disease in Black postmenopausal females.

Fibrinolytic treatment using recombinant tissue-type plasminogen activator (rt-PA) for staphylococcal infective endocarditis.

Infective endocarditis (IE) is a severe illness characterized by vegetation of bacterial thrombosis. We hypothesized that adding recombinant tissue-type plasminogen activator (rt-PA) to antibiotics would contribute to good results in the treatment of IE. As an in vitro study, we injected labeled Staphylococcus aureus (S. aureus) and either rt-PA or PBS + plasminogen into a polydimethylsiloxane flow chamber with fibrin on a coverslip, and then performed immunofluorescent area assessment. As an in vivo experiment, IE model rats that had suffered mechanical damage in the aortic valve by catheter and revealed bacterial vegetation caused by S. aureus injection were treated with either a control, cefazolin (CEZ), rt-PA, or rt-PA + CEZ, for 7 days. Survival was assessed for 14 days after the appearance of vegetation, with daily monitoring of the vegetation by transthoracic echocardiography (TTE). The in vitro investigation showed that perfusion of rt-PA could detach S. aureus significantly more efficiently than PBS could. In the in vivo research, the rt-PA + CEZ group survived significantly longer than the other groups, and rt-PA + CEZ was more effective than CEZ in the dissolution of vegetation, as observed by TTE. In conclusion, adding rt-PA to antibiotic treatment could dissolve the vegetation component synergistically and improve the survival rate.

RUNX1 targeting AKT3 promotes alveolar hypercoagulation and fibrinolytic inhibition in LPS induced ARDS.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are mainly responsible for massive alveolar fibrin deposition, which are closely related with refractory hypoxemia in acute respiratory distress syndrome (ARDS). Our previous study testified runt-related transcription factor (RUNX1) participated in the regulation of this pathophysiology in this syndrome, but the mechanism is unknown. We speculate that screening the downstream genes associated with RUNX1 will presumably help uncover the mechanism of RUNX1. METHODS: Genes associated with RUNX1 were screened by CHIP-seq, among which the target gene was verified by Dual Luciferase experiment. Then the efficacy of the target gene on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS was explored in vivo as well as in vitro. Finally, whether the regulatory effects of RUNX1 on alveolar hypercoagulation and fibrinolytic in ARDS would be related with the screened target gene was also sufficiently explored. RESULTS: Among these screened genes, AKT3 was verified to be the direct target gene of RUNX1. Results showed that AKT3 was highly expressed either in lung tissues of LPS-induced rat ARDS or in LPS-treated alveolar epithelia cell type II (AECII). Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) were increasingly expressed both in lung tissues of ARDS and in LPS-induced AECII, which were all significantly attenuated by down-regulation of AKT3. Inhibition of AKT3 gene obviously ameliorated the LPS-induced lung injury as well as the collagen I expression in ARDS. RUNX1 overexpression not only promoted the expressions of TF, PAI-1, but also boosted AKT3 expression in vitro. More importantly, the efficacy of RUNX1 on TF, PAI-1 were all effectively reversed by down-regulation of AKT3 gene. CONCLUSION: AKT3 is an important target gene of RUNX1, through which RUNX1 exerted its regulatory role on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. RUNX1/ATK3 signaling axis is expected to be a new target for the exploration of ARDS genesis and treatment.

Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Biomolecular characterization of Antheraea mylitta cocoonase: A secreted protease.

Cocoonase is a protease secreted during the emergence of silk moths. In the present study cocoonase of Antheraea mylitta was collected, purified and secondary structure was determined using circular dichroism (CD) spectroscopy which revealed the presence of α-helix 4.3%, ß-sheet 55%, turn 8% and random coil 32.7%. The thermal stability of cocoonase was studied using CD spectroscopy while the thermal property was observed using Differential Scanning Calorimetry (DSC). Furthermore, MALDI-TOF peptide mass fingerprinting (PMF) was performed for similar protein identification using the MASCOT server. Using casein as the substrate, the kinetic constants Km and Vmax were 13 × 103 mg/ml and 15.09 × 10-2 µg/mg.s1 respectively. The specific activity of cocoonase was observed to be maximum at temperature 40 °C, pH-8.0. The effect of heavy metals Hg2+, Cd2+, Co2+, Pb2+ showed inhibitory activity at higher concentrations, while few metals like Mn2+, Fe3+ enhanced the activity while the effect of Ca2+ was not much on the activity. Soybean trypsin inhibitor and PMSF showed an inhibitory effect on the activity of cocoonase. Additionally, antioxidant scavenging and fibrinolytic properties were also observed. Furthermore, the imperative information generated through the present study will serve to explore cocoonase for its prospective pharmaceutical applications.

Heterologous expression, purification and single step efficient refolding of recombinant tissue plasminogen activator (Reteplase) from E. coli.

Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) E. coli expression vector and expressed in Rosetta-gami 2 E. coli (NovagenⓇ) host. rPA was expressed as an inclusion body in E. coli and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and In Vitro clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield.

Irrigation with fibrinolytic agents versus saline for percutaneous drainage of abdominal abscesses: A meta-analysis with trial sequential analysis of randomized trials.

INTRODUCTION: Fibrinolytic agents (FA) activate the fibrinolytic system, converting plasminogen into plasmin to break down fibrin. Their use for irrigation of abdominal abscesses is debated, and this meta-analysis evaluates their efficacy. METHODS: We searched PubMed, Embase, and Cochrane Central for randomized controlled trials (RCTs) comparing FA and saline in percutaneous drainage of abdominal abscesses. Outcomes included length of hospitalization, duration of drainage, and drainage volume. We pooled mean differences (MD) and 95% confidence intervals (CI) using a random-effects model. We also performed a trial sequential analysis (TSA). RESULTS: We included six RCTs encompassing 299 patients. In the overall analysis, FA increased drainage volume (MD 104.25 mL; 95% CI 35.72-172.77 mL; p = 0.003; I2 = 0%). In children, saline reduced hospitalization duration (MD -1.26 days; 95% CI -1.98 to -0.55 days; p = 0.0006; I2 = 0%), whereas FA increased drainage volume (MD 84.66 mL; 95% CI 5.77-153.54 mL; p = 0.04; I2 = 0%). In adults, FA significantly reduced hospitalization duration (MD -11.12 days; 95% CI -15.16 to -7.08 days; p < 0.00001; I2 = 0%) and duration of drainage (MD -6.53 days; 95% CI -9.25 to -3.81 days; p < 0.00001; I2 = 0%) while increasing drainage volume (MD 164.47 mL; 95% CI 26.16-302.78 mL; p = 0.02; I2 = 0%). On TSA, the required information size was achieved only for the adult subgroup's hospitalization and drainage duration. CONCLUSION: In adults, FA reduce hospitalization and drainage duration and increase drainage volume. In children, saline seems more effective in reducing hospitalization duration, while FA increase drainage volume. These findings underscore the need for age-specific treatments and further research, especially in the pediatric population.

Spontaneous intracerebral hemorrhage - patients retrospectively consent to fibrinolytic surgery despite poor neurological outcome and reduced health-related quality of life.

Spontaneous intracerebral hemorrhage (ICH) might lead to devastating consequences. Nonetheless, subjective interpretation of life circumstances might vary. Recent data from ischemic stroke patients show that there might be a paradox between clinically rated neurological outcome and self-reported satisfaction with quality of life. Our hypothesis was that minimally invasive surgically treated ICH patients would still give their consent to stereotactic fibrinolysis despite experiencing relatively poor neurological outcome. In order to better understand the patients' perspective and to enhance insight beyond functional outcome, this is the first study assessing disease-specific health-related quality of life (hrQoL) in ICH after fibrinolytic therapy. We conducted a retrospective analysis of patients with spontaneous ICH treated minimally invasive by stereotactic fibrinolysis. Subsequently, using standardized telephone interviews, we evaluated functional outcome with the modified Rankin Scale (mRS), health-related Quality of Life with the Quality of life after Brain Injury Overall scale (QOLIBRI-OS), and assessed retrospectively if the patients would have given their consent to the treatment. To verify the primary hypothesis that fibrinolytic treated ICH patients would still retrospectively consent to fibrinolytic therapy despite a relatively poor neurological outcome, we conducted a chi-square test to compare good versus poor outcome (mRS) between consenters and non-consenters. To investigate the association between hrQoL (QOLIBRI-OS) and consent, we conducted a Mann-Whitney U-test. Moreover, we did a Spearman correlation to investigate the correlation between functional outcome (mRS) and hrQoL (QOLIBRI-OS). The analysis comprised 63 data sets (35 men, mean age: 66.9 ± 11.8 years, median Hemphill score: 3 [2-3]). Good neurological outcome (mRS 0-3) was achieved in 52% (33/63) of the patients. Patients would have given their consent to surgery retrospectively in 89.7% (52/58). These 52 consenting patients comprised all 33 patients (100%) who achieved good functional outcome and 19 of the 25 patients (76%) who achieved poor neurological outcome (mRS 4-6). The mean QOLIBRI-OS value was 49.55 ± 27.75. A significant association between hrQoL and retrospective consent was found (p = 0.004). This study supports fibrinolytic treatment of ICH even in cases when poor neurological outcome would have to be assumed since subjective perception of deficits could be in contrast with the objectively measured neurological outcome. HrQoL serves as a criterion for success of rtPa lysis therapy in ICH.

Timeliness of reperfusion in ST-segment elevation myocardial infarction and outcomes in Kerala, India: results of the TRUST outcomes registry.

PURPOSE: Transatlantic guidelines endorse quality metrics for timely reperfusion in patients with ST-elevation myocardial infarction (STEMI). Compliance in low- and middle-income countries (LMICs) is largely unknown. STUDY DESIGN: We prospectively evaluated 2928 STEMI patients in Kerala, India, across 16 PCI-capable hospitals who received reperfusion with either primary percutaneous coronary intervention (PPCI) or fibrinolysis. Primary endpoint was a major adverse cardiovascular event (MACE) composite of death, non-fatal myocardial infarction, stroke or readmission for heart failure at 1-year. RESULTS: Among reperfused STEMI patients, 320 (10.9%) received timely reperfusion with either PPCI or fibrinolysis, 1985 (67.8%) received delayed PPCI, and 623 (21.3%) received delayed fibrinolysis. Timely reperfusion had lower unadjusted MACE rates than delayed PCI or fibrinolysis (timely reperfusion: 11.9%, delayed PPCI: 13.6%, delayed fibrinolysis: 23.9%, P < 0.001). Mortality was lowest in the timely reperfusion group (timely reperfusion: 6.3%, delayed PPCI: 7.8%, delayed fibrinolysis 18.8%, P < 0.001). After multivariate analysis, delayed fibrinolysis had a higher MACE rate (HR 1.52 95% CI 1.04-2.21) and mortality (HR 1.97, 95% CI 1.18-3.25) compared to timely reperfusion. Total ischemic time > 3 h and delayed first medical contact-to-needle time predicted MACE at 1 year. CONCLUSIONS: Among STEMI patients in Kerala, India, only one in 10 eligible patients received timely reperfusion. Longer total ischemic times and delayed fibrinolysis were associated with 1-year MACE. Improving timely reperfusion is critical to enhancing STEMI outcomes in LMICs. What is already known on this topic Given the established link between delay to reperfusion and worse major adverse cardiac events (MACE), global efforts have concentrated on minimizing different components of the total ischemic time to improve ST-elevation myocardial infarction (STEMI) outcomes. Compliance in low- and middle-income countries (LMICs) is largely unknown. What this study adds In this cohort of STEMI patients in Kerala, India, total ischemic time and first medical contact-to-needle time correlated with long-term MACE rates, whereas other timeliness indicators did not. How this study might affect research, practice or policy Our study highlights the significant barriers to accessing STEMI care that are prevalent in LMICs despite incremental growth in the number of PCI-capable hospitals. The pre-hospital phase within total ischemic time is the most important quality improvement metric of STEMI care in LMICs, especially for patients chosen for fibrinolysis.

Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea.

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 µM and 49.0 µmol min-1 mg-1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Protection of Tight Junctional Complexes between hCMEC/D3 Cells by Deep-Sea Fibrinolytic Compound FGFC1.

Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood-brain barrier (BBB), whose regulation closely correlates to the BBB's integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of the BBB via the construction of an in vitro model. This study aims to investigate the protective effect of the deep-sea-derived fibrinolytic compound FGFC1 against H2O2-induced dysfunction of TJCs and to elucidate the underlying mechanism. The barrier function was shown to decline following exposure to 1 mM H2O2 in an in vitro model of hCMEC/D3 cells, with a decreasing temperature-corrected transendothelial electrical resistance (tcTEER) value. The decrease in the tcTEER value was significantly inhibited by 80 or 100 µM FGFC1, which suggested it efficiently protected the barrier integrity, allowing it to maintain its function against the H2O2-induced dysfunction. According to immunofluorescence microscopy (IFM) and quantitative real-time polymerase chain reaction (qRT-PCR), compared to the H2O2-treated group, 80~100 µM FGFC1 enhanced the expression of claudin-5 (CLDN-5) and VE-cadherin (VE-cad). And this enhancement was indicated to be mainly achieved by both up-regulation of CLDN-5 and inhibition of the down-regulation by H2O2 of VE-cad at the transcriptional level. Supported by FGFC1's molecular docking to these proteins with reasonable binding energy, FGFC1 was proved to exert a positive effect on TJCs' barrier function in hCMEC/D3 cells via targeting CLDN-5 and VE-cad. This is the first report on the protection against H2O2-induced barrier dysfunction by FGFC1 in addition to its thrombolytic effect. With CLDN-5 and VE-cad as the potential target proteins of FGFC1, this study provides evidence at the cellular and molecular levels for FGFC1's reducing the risk of bleeding transformation following its application in thrombolytic therapy for cerebral thrombosis.

Establishing reliable selection criteria for performing fibrinolytic therapy in patients with intracerebral haemorrhage based on prognostic tools.

OBJECTIVES: Minimally invasive surgery combined with fibrinolytic therapy is a promising treatment option for patients with intracerebral haemorrhage (ICH), but a meticulous patient selection is required, because not every patient benefits from it. The ICH score facilitates a reliable patient selection for fibrinolytic therapy except for ICH-4. This study evaluated whether an additional use of other prognostic tools can overcome this limitation. MATERIALS AND METHODS: A consecutive ICH patient cohort treated with fibrinolytic therapy between 2010 and 2020 was retrospectively analysed. The following prognostic tools were calculated: APACHE II, ICH-GS, ICH-FUNC, and ICH score. The discrimination power of every score was determined by ROC-analysis. Primary outcome parameters regarding the benefit of fibrinolytic therapy were the in-hospital mortality and a poor outcome defined as modified Rankin scale (mRS) > 4. RESULTS: A total of 280 patients with a median age of 72 years were included. The mortality rates according to the ICH score were ICH-0 = 0 % (0/0), ICH-1 = 0 % (0/22), ICH-2 = 7.1 % (5/70), ICH-3 = 17.3 % (19/110), ICH-4 = 67.2 % (45/67), ICH-5 = 100 % (11/11). The APACHE II showed the best discrimination power for in-hospital mortality (AUC = 0.87, p < 0.0001) and for poor outcome (AUC = 0.79, p < 0.0001). In the subgroup with ICH-4, APACHE II with a cut-off of 24.5 showed a good discriminating power for in-hospital mortality (AUC = 0.83, p < 0.001) and for poor outcome (AUC = 0.87, p < 0.001). CONCLUSIONS: An additional application of APACHE II score increases the discriminating power of ICH score 4 enabling a more precise appraisal of in-hospital mortality and of functional outcome, which could support the patient selection for fibrinolytic therapy.

Recovery and partial purification of fibrinolytic protease from Pleurotus ostreatus and P. eryngii and cytotoxic and antioxidant activity of their extracts.

Mushrooms are a source of primary and secondary metabolites. Little is known about the most suitable conditions for production of mushrooms by submerged fermentation. This article reports antioxidant and cytotoxic assays, in addition to quantitatively evaluating the content of proteases with fibrinolytic action in the crude extracts of two species of edible mushrooms produced in different formulations, as well as evaluating the recovery of these enzymes by aqueous two-phase systems (ATPS). The mushrooms Pleurotus ostreatus and Pleurotus eryngii, at concentration of 100 µg/mL, displayed inhibition of DPPH and ABTS radicals below 50%. In the cytotoxicity test, the cells human fibroblast cell lines (MRC-5) showed cell viability greater than 80%. Concerning fibrinolytic activity, P. eryngii presented 226.47 ± 7.26 U/mL, therefore being more efficient than P. ostreatus (71.5 ± 0.56 U/mL). In the recovery of the P. eryngii extract by ATPS, the fibrinolytic protease was partitioned in the salt phase (30.25 U/mL). The molecular mass of the proteases was between 75 and 100 kDa. These results prove the low cytotoxicity of the extracts produced and that fermentation in supplemented malt broth favored the excretion of fibrinolytic proteases compared to the other evaluated media.

Empirical and modified hemostatic resuscitation for liver blast injury combined with seawater immersion: A preliminary study.

PURPOSE: To compare the effects of empirical and modified hemostatic resuscitation for liver blast injury combined with seawater immersion. METHODS: Thirty rabbits were subjected to liver blast injury combined with seawater immersion, and were then divided into 3 groups randomly (n = 10 each): group A (no treatment after immersion), group B (empirical resuscitation with 20 mL hydroxyethyl starch, 50 mg tranexamic acid, 25 IU prothrombin complex concentrate and 50 mg/kg body weight fibrinogen concentrate), and group C (modified resuscitation with additional 10 IU prothrombin complex concentrate and 20 mg/kg body weight fibrinogen concentrate based on group B). Blood samples were gathered at specified moments for assessment of thromboelastography, routine coagulation test, and biochemistry. Mean arterial pressure, heart rate, and survival rate were also documented at each time point. The Kolmogorov-Smirnov test was used to examine the normality of data distribution. Multigroup comparisons were conducted with one-way ANOVA. RESULTS: Liver blast injury combined with seawater immersion resulted in severe coagulo-fibrinolytic derangement as indicated by prolonged prothrombin time (s) (11.53 ± 0.98 vs. 7.61 ± 0.28, p＜0.001), activated partial thromboplastin time (APTT) (s) (33.48 ± 6.66 vs. 18.23 ± 0.89, p＜0.001), reaction time (R) (min) (5.85 ± 0.96 vs. 2.47 ± 0.53, p＜0.001), decreased maximum amplitude (MA) (mm) (53.20 ± 5.99 vs. 74.92 ± 5.76, p＜0.001) and fibrinogen concentration (g/L) (1.188 ± 0.29 vs. 1.890 ± 0.32, p = 0.003), and increased D-dimer concentration (mg/L) (0.379 ± 0.32 vs. 0.051 ± 0.03, p = 0.005). Both empirical and modified hemostatic resuscitation could improve the coagulo-fibrinolytic states and organ function, as indicated by shortened APTT and R values, decreased D-dimer concentration, increased fibrinogen concentration and MA values, lower concentration of blood urea nitrogen and creatine kinase-MB in group B and group C rabbits in comparison to that observed in group A. Further analysis found that the R values (min) (4.67 ± 0.84 vs. 3.66 ± 0.98, p = 0.038), APTT (s) (23.16 ± 2.75 vs. 18.94 ± 1.05, p = 0.001), MA (mm) (60.10 ± 4.74 vs. 70.21 ± 3.01, p < 0.001), and fibrinogen concentration (g/L) (1.675 ± 0.21 vs. 1.937 ± 0.16, p = 0.013) were remarkably improved in group C than in group B at 2 h and 4 h after injury. In addition, the concentration of blood urea nitrogen (mmol/L) (24.11 ± 1.96 vs. 21.00 ± 3.78, p = 0.047) and creatine kinase-MB (U/L) (85.50 ± 13.60 vs. 69.74 ± 8.56, p = 0.013) were lower in group C than in group B at 6 h after injury. The survival rates in group B and group C were significantly higher than those in group A at 4 h and 6 h after injury (p < 0.001), however, there were no statistical differences in survival rates between group B and group C at each time point. CONCLUSIONS: Modified hemostatic resuscitation could improve the coagulation parameters and organ function better than empirical hemostatic resuscitation.

Oral fibrinolytic agent in hyper-coagulopathy in severe COVID - 19 ARDS Patients: two case reports.

Abstract: The use of oral fibrinolytic agent (DLBS1033) has been proven for adjuvant treatment in venous thromboembolism, however until now there is no published report about its uses and effectiveness as an addition to the standard therapy of severe COVID-19 cases and hypercoagulopathy. We present two cases of severe confirmed COVID-19 from PCR tests, seen at Ngimbang Hospital, Lamongan, East Java in October and November, 2020. The first patient was a 51-year-old male who presented to ER with fever, dyspnoea, cough, and oxygen desaturation (SpO2 room air 87%), with comorbids of pulmonary hypertension (PH), atrial fibrillation, heart failure secondary to corpulmonale, and hypercoagulopathy. The second patient was a 56-yearold female who presented with fever, dyspnoea, and oxygen desaturation (Sp02 room air 88%), with comorbid ARDS, hypertension, hyperglycaemia, hypercoagulopathy, heart failure, and CAD. Both of the patients were treated with standard treatment therapy for severe COVID-19 and comorbid therapy, and DLBS1033 in addition to fondaparinux due to limited hospital resources. Both patients showed good clinical outcomes after the course of treatment and had no adverse effects. CONCLUSIONS: Our two case reports were the first that showed good clinical outcome and safety of DLBS1033 treatment in addition to fondaparinux for hypercoagulopathy therapy.

Tenecteplase in Acute Stroke: What About the Children?

Tenecteplase is replacing alteplase as the fibrinolytic agent of choice for the acute management of ischemic stroke in many adult stroke centers due to practical and pharmacokinetic advantages in the setting of similar outcomes. Although thrombolytic use is increasing for acute childhood stroke, there is very limited experience with tenecteplase in children for any indication, and importantly, there are no data on safety, dosing, or efficacy of tenecteplase for childhood stroke. Changes in fibrinolytic capacity over childhood, pediatric pharmacological considerations such as age-specific differences in drug clearance and volume of distribution, and practical aspects of drug delivery such as availability in children's hospitals may impact decisions about transitioning from alteplase to tenecteplase for acute pediatric stroke treatment. Pediatric and adult neurologists should prepare institution-specific guidelines and organize prospective data collection.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus.

BACKGROUND: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component. METHODS: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions. RESULTS: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes. CONCLUSION: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

Isolation, identification, and optimization of the fibrinolytic protease-producing strains.

Cardiovascular complications due to thrombosis have become one of the main causes of death worldwide. The high cost and undesirable side effects of existing thrombolytic agents have led researchers to isolate potential strains that produce fibrinolytic enzymes for therapeutic applications. Fibrinolytic enzymes, especially of microbial origin, are recognized as potential therapeutic candidates for thrombosis. In this study, isolation, identification, and optimization of fibrinolytic protease enzyme-producing strains were performed using fermentative protein sources. Fibrinolytic protease-producing strains were selected by analyzing the isolated strains on skim milk agar medium. The selected strains were examined on blood agar and fibrin plate medium, and the ones showing high enzymatic activity were determined. The strain determined to have the highest activity was identified as Acinetobacter johnsonii TR01 by 16S rRNA analysis. The maximum fibrinolytic protease production of the strain occurred at 60 °C and pH 7.0. Under different medium conditions used for enzyme production, fructose was found to be the best carbon source, while yeast extract and peptone were the best nitrogen sources. It was observed that CaCl2, KH2PO4, and MgSO4 components had a negative effect, while MnCl2 and ZnC4H6O4 components had a positive effect on enzyme production. The medium composition for maximum enzyme activity (8.30 IU/ml) determined by Response Surface Methodology was 14.22 g/L fructose, 11.190 g/L yeast extract, 14.22 g/L peptone, 0.5 g/L MnCl2, and 0.5 g/L ZnC4H6O4.